Development and validation of a competitive enzyme-linked immunosorbent assay for the measurement of total plasma immunoglobulins in healthy loggerhead sea (Caretta caretta) and green turtles (Chelonia mydas).

The quantification of circulating plasma immunoglobulins represents a valuable diagnostic tool in human and veterinary immunology, although its application is very limited in reptile medicine to date. The objectives of our study were the development and standardization of a competitive enzyme-linked immunosorbent assay (cELISA) for the measurement of total plasma immunoglobulins (Igs; both IgM and IgY) in loggerhead sea turtles (LST; Caretta caretta; n = 254) and green turtles (GT; Chelonia mydas; n = 111), the establishment of reference intervals for Ig for both species, and the examination of associations between Ig and total protein (TP), condition index, and water temperature. The cELISA for Ig was successfully developed and optimized. Reference intervals for Ig were 0.38-0.94 g/dL in LST (median: 0.59 g/dL; range: 0.16-2.15 g/dL) and 0.40-0.85 g/dL in GT (median: 0.58 g/dL; range: 0.18-1.80 g/dL). In LST, there were positive linear relationships of Ig with TP, and TP with Ig and condition index, and a negative relationship of Ig with condition index. The positive linear relationships of Ig with TP, and TP with Ig were also identified in GT. These positive associations of Ig and TP were expected, as Ig represents fractions of TP, and TP reportedly increases with straight carapace length and weight. The negative association of Ig with condition index may indicate potential biological variations. The cELISA and reference intervals for total Ig of LST and GT presented herein have the potential to be useful as a diagnostic and research tool for sea turtle immunology.

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus).

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.